Total synthesis of the Marine cyclic depsipeptide viequeamide A

Article abstract of DOI:10.1021/np4001027

The first total synthesis of viequeamide A, a natural cyclic depsipeptide isolated from a marine button cyanobacterium, was achieved with the N-Me-Val-Thr peptide bond as the final macrocyclization site. The synthetic product gave nearly identical spectroscopic data to that reported for the natural product.

Full text of DOI:10.1021/np4001027

Note
pubs.acs.org/jnp
Total Synthesis of the Marine Cyclic Depsipeptide Viequeamide A
Dongyu Wang,†,‡ Shanshan Song,§ Ye Tian,‡ Youjun Xu,*,† Zehong Miao,§ and Ao Zhang*,‡
†School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, People’s Republic of China
‡CAS Key Laboratory of Receptor Research, and Synthetic Organic & Medicinal Chemistry Laboratory (SOMCL), Shanghai Institute
of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai, People’s Republic of China
§State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai,
People’s Republic of China
S
* Supporting Information
ABSTRACT: The first total synthesis of viequeamide A, a
natural cyclic depsipeptide isolated from a marine button
cyanobacterium, was achieved with the N-Me-Val−Thr
peptide bond as the final macrocyclization site. The synthetic
product gave nearly identical spectroscopic data to that
reported for the natural product.
three fragments, the tripeptide P1, dipeptide P2, and ester P3,
based on the retrosynthetic analysis as outlined in Figure 1.
The tripeptide fragment P1 consists of the Hmpa−N-Me-
Val−Val amino acid sequence. Its synthesis began with the
conversion of L-isoleucine to (2R,3S)-2-hydroxy-3-methylpen-
tanoic acid (2R,3S-Hmpa). As shown in Scheme 1,
diazotization of L-isoleucine with NaNO2, followed by
protection of the acid with allyl bromide, provided allyl ester
34 in 62% overall yield. Condensation of ester 3 with N-methyl-
N-Boc-L-valine7 following a modified Mitsunobu procedure
produced the epimerized dipeptide 45 in 91% yield. Removal of
the N-Boc protecting group in 4 by treating with TFA, followed
by condensation with N-Boc-protected L-valine under the
typical peptide coupling conditions3,6 (HATU/HOAt/
(iPr)2NEt), afforded fragment P1 in 66% overall yield with
iequeamide A (1) is a natural cyclic depsipeptide recently
1
Visolated by Gerwick and co-workers
from a marine
“button” cyanobacterium (Rivularia sp.) near the island of
Vieques, Puerto Rico. Structurally, it belongs to the kulolide
superfamily featuring a (3S)-2,2-dimethyl-3-hydroxy-7-octynoic
acid (3S-Dhoya) moiety with divergent biological activities.2
Viequeamide A is a 22-membered macrocycle comprising nine
stereogenic centers. In addition to the 3S-Dhoya moiety, it
contains six other amino/hydroxy acid fragments, including one
L-proline (L-Pro), one L-valine (L-Val), one L-threonine (L-Thr),
two N-methylated-L-valines (L-N-MeVal), and one (2R,3S)-2-
hydroxy-3-methylpentanoic acid (2R,3S-Hmpa) (Figure 1).
More attractively, this natural product was found to be highly
toxic against H460 human lung cancer cell lines, with an IC50
value of 60 nM,1 strikingly different from other related peptides
isolated from the same natural source, which were shown to be
inactive to the same cell lines. The structure and significant
cytotoxicity made viequeamide A (1) an optimal target for total
synthesis and further biological activity screening.
1
no epimerization according to the H and 13C NMR analyses.
Preparation of the dipeptide fragment P2 has been reported
earlier by our group3 (Scheme 1). It should be mentioned that
N-methylation of N-Boc-L-valine made both compounds 5 and
P2 exist as a mixture of amide rotamers due to steric hindrance.
Our group recently prepared the diastereomer of P2 starting
from N-Boc-D-valine and ruled out racemization of P2 during
amide bond formation.3
The synthesis of ester fragment P3 is outlined in Scheme 2.
First, the commercially available hex-5-yn-1-ol was oxidized8 to
hex-5-ynal with NaOCl/TEMPO in 70% yield. Subsequent
aldol condensation3,9 of hex-5-ynal with chiral amide 9R
Although there are several options to retrosynthetically
dissect this molecule, our experience with the synthesis of the
related peptide veraguamide A3 suggested that the esterification
between the L-Thr and 3S-Dhoya moieties might be trouble-
some due to the steric hindrance of the highly methylated
Dhoya component. Therefore, this coupling needs to be
established earlier in the synthesis. Meanwhile, formation of the
N-MeVal−Thr peptide bond seems optimal for the final
macrocyclization in view of the reduced steric hindrance of
these two amino acid moieties relative to other residues in the
molecule. In this regard, viequeamide A was disconnected into
Received: February 3, 2013
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Note
Figure 1. Structure of viequeamide A and our retrosynthetic analysis.
asymmetric Evans’ aldol synthetic strategy10 reported by
Gerwick et al., 5-hexynal reacted with 9S in the presence of
LDA and Ti(O-iPr)3Cl to provide the aldol adduct 6 in 61%
yield. Removal of the Evans’ chiral auxiliary using H2O2/LiOH
a
Scheme 1. Synthesis of Fragments P1 and P2
provided 3S-Dhoya (7) in 95% yield ([α]25 −24.3 (c 0.4,
D
CHCl3), lit.10 −26.3 (c 1.0, CHCl3)).
Esterification of acid 7 with allyl bromide afforded allyl ester
8 in 93% yield (Scheme 2). As expected, the subsequent
condensation of ester 8 with L-threonine (L-Thr) proved to be
challenging due to steric hindrance. After attempting various
conditions with L-Thr substrates (Table 1), we found that the
Table 1. Acylation of Alcohol 8
entry
1
conditions
yield
Fmoc-L-Thr(tBu)-Cl (5.0 equiv), DMAP (0.5 equiv), iPr2NEt trace
(6.0 equiv), CH2Cl2
a
Reaction condition and reagents: (i) NaNO2, H2SO4; (ii) allyl
2
3
Fmoc-L-Thr(tBu)−OH (2.0 equiv), DCC (3.0 equiv), 4-PPY 33%
bromide, K2CO3,TBAB; (iii) N-Me-N-Boc-L-Val, DEAD, PPh3, 0 °C
to rt; (iv) TFA, CH2Cl2; (v) N-Boc-L-Val, HATU, HOAt, DIPEA; (vi)
MeI, NaH, THF; (vii) N-Boc-L-proline, HATU, HOAt, DIPEA.
(3.0 equiv), CH2Cl2
Fmoc-L-Thr(tBu)−OH (2.0 equiv), 2,4,6-trichlorobenzoyl
chloride (2.0 equiv), DMAP (2.0 equiv), iPr2NEt (2.5
equiv)
48%
a
Scheme 2. Synthesis of Fragment P3
4
5
Fmoc-L-Thr(tBu)−OH (2.0 equiv), EDCI (3.0 equiv),
52%
81%
DMAP (3.0 equiv), CH2Cl2
Fmoc-L-Thr(tBu)−OH (5.0 equiv), EDCI (10.0 equiv),
DMAP (10.0 equiv), CH2Cl2
best result could be achieved by using Fmoc-L-Thr(tBu)−OH
(5 equiv), EDCI (10 equiv), and DMAP (10 equiv), leading to
the P3 fragment in 81% yield with no epimerization according
1
to the H and 13C NMR analysis.
With the three key fragments (P1, P2, P3) in hand, we were
ready to assemble precursor 2 by choosing the formation of the
N-MeVal−Thr (P2−P3) amide bond as the final macro-
cyclization step. Accordingly, the connecting strategy P2 → P1
→ P3 → P2 was initiated.
a
Reaction condition and reagents: (i) NaOCl, TEMPO, NaBr; (ii) 9,
As described in Scheme 3, removal of the O-allyl group in the
P1 fragment with Pd(PPh3)4 at 0 °C afforded acid 10 in 85%
yield.3 Meanwhile, removal of N-Boc protection in P2 by
treating with TFA followed by condensation with acid 10 under
the standard peptide bond-forming conditions3,6 (HATU/
HOAt/DIPEA at 0 °C) yielded product 12 in 60% overall yield
(two steps). In order to prepare fragment 14, we initially
LDA, Ti(O-iPr)3Cl, THF, −78 °C, then hex-5-ynal, −40 °C; (iii)
H2O2, LiOH, THF/H2O; (iv) allyl bromide, KHCO3, DMF; (v)
Fmoc-L-Thr(tBu)−OH, EDCI, DMAP.
(prepared by methylation of (R)-4-benzyl-3-propionyloxazoli-
din-2-one9) under the standard conditions (Bu2BOTf/Et3N at
−78 °C) was unsuccessful. Fortunately, by following a modified
B
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Note
a
Scheme 3. Final Assembly of Viequeamide A (1)
Figure 2. Comparison of 1H and 13C NMR spectra for synthetic 1 and
natural viequeamide A.
synthetic compound. All the comparisons secured the total
synthesis of viequeamide A.
The cytotoxicity of synthetic 1, as well as paclitaxel and
etoposide (as positive controls), was evaluated against several
cell lines,14 including human lung cancer A549 cells, squamous
cell carcinoma KB cells, vincristine-resistant KB/VCR cells, and
human lung cancer H460 cells. Unfortunately, compared to the
high potency of paclitaxel (3.2 nM) and etoposide (63.1 nM) in
our assays,14 synthetic compound 1 was found to be inactive in
all four cancer cells, with IC50 values up to 100 μM. HPLC
analysis of the bioassay sample (in DMSO solution) indicated
that compound 1was unstable and decomposed to several side
products. Although more investigations are needed, the
instability of synthetic 1 may be a partial rationale for its
poor cytotoxicity. Further studies on the side products are
ongoing.
a
Reaction condition and reagents: (i) Pd(PPh3)4, N-methylaniline; (ii)
TFA, CH2Cl2; (iii) HATU, HOAt, DIPEA; (iv) Fmoc-L-Thr(tBu)−
OH, EDCI, DMAP; (v) Et2NH, CH3CN.
CONCLUSIONS
■
conducted a stepwise procedure by directly using 3S-Dhoya (7)
instead of P3 to minimize the steric effects. Removal of N-Boc
protection of 12 provided the primary amine intermediate,
which was then condensed with acid 7. The reaction went
smoothly, and product 13 was obtained in 63% overall yield
(two steps). However, esterification of 13 with Fmoc-L-
Thr(tBu)−OH under the same conditions as used for the
preparation of P3 did not occur.
In summary, we have achieved the first total synthesis of
viequeamide A, a natural cyclic depsipeptide isolated from a
marine button cyanobacterium, through 10 linear steps based
on the three retrosynthetic fragments with 0.9% overall yield.
Our synthesis formed the N-MeVal−Thr peptide bond as the
final macrocyclization site, and all of the spectroscopic data of
the synthetic product were in good agreement with those
reported for the natural product.
Alternatively, removal of the allyl protecting group in P3 with
a palladium catalyst (Pd(PPh3)4/N-methylaniline) yielded the
corresponding acid intermediate, which was condensed with
the liberated primary amine from pentapeptide 12 under the
standard coupling conditions.3,6 Amide 14 was obtained in 68%
overall yield. After sequential removal of the allyl group with
Pd(PPh3)4/N-methylaniline to liberate the acid function and
removal of the Fmoc group by diethylamine to release the
amino group in 14, the product was subjected to macro-
cyclization under the standard conditions to generate the key
cyclic peptide 2 in 41% yield (three steps).11,12 Finally,
treatment of precursor 2 with 20% TFA in CH2Cl2 furnished
the expected target 1 in 88% yield.
EXPERIMENTAL SECTION
■
General Experimental Procedures. All reactions were per-
formed in glassware containing a Teflon-coated stir bar. CH2Cl2 and
THF were purified and dried according to the standard methods prior
to use. All reagents were obtained from commercial sources and used
without further purifications. NMR spectra were recorded on Varian-
1
MERCURY Plus-300 (300 MHz for H), Variant MR-400 (100 MHz
for 13C), and AVANCE III 500 (125 MHz for 13C). 1H and 13C NMR
spectra were recorded with tetramethylsilane as an internal reference.
Low- and high-resolution mass spectra were obtained on Finni-
ganLTQ and Micromass Ultra Q-TOF in the ESI mode. Optical
rotations were recorded with a Perkin-Elmer 341MC polarimeter.
Flash column chromatography on silica gel (200−300 mesh) was used
for the routine purification of reaction products, and a mixture of
EtOAc and petroleum ether was used as the eluent. The column
output was monitored by TLC on silica gel (100−200 mesh)
precoated on glass plates (10 cm × 50 cm), and spots were visualized
by UV light at 254 nM or by potassium permanganate color agent.
(2R,3S)-Allyl-2-((S)-2-(tert-butoxycarbonyl(methyl)amino)-3-
methylbutanoyloxy)-3-methylpentanoate (4). A solution of
1
As shown in Figure 2, both the H and 13C NMR spectra of
our synthetic product 1 were in good agreement with those
reported for the natural product viequeamide A.1 Meanwhile,
our synthetic product 1 showed a specific rotation [α]20 of
D
−34.7 (c 0.15, CH2Cl2), also comparable to that for the natural
product:1 −32.6 (CH2Cl2). In addition, a direct HPLC analysis
with an authentic sample13 also confirmed the identity of our
C
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Note
diethyl azodicarboxylate (9.96 mmol) in toluene (4.5 mL) was added
to a solution of 3 (570 mg, 3.32 mmol), triphenylphosphine (2.9
g,14.94 mmol), and N-methyl-N-Boc-valine (2.3 g, 9.96 mmol) in
THF (30 mL) at 0 °C. The resulting solution was stirred at rt for 14 h.
The solvent was removed under reduced pressure and then diluted
with H2O. After extraction with EtOAc, the combined organic phase
was washed with brine and dried with Na2SO4. Removal of the
solvents followed by flash chromatography (petroleum ether/CH2Cl2,
3:2) provided 4 as a colorless oil (1.16 g, 91%): 1H NMR (300 MHz,
CDCl3) δ 5.94−5.81 (1H, m), 5.27 (2H, dd, J = 22.7, 13.8 Hz), 5.01
(1H, s), 4.62 (2H, d, J = 5.8 Hz), 4.43 (1H, m), 2.83 (3H, d, J = 11.9
Hz), 2.27−2.12 (1H, m), 2.08−1.92 (1H, m), 1.45 (9H, s), 1.38−1.19
(2H, m), 1.05−0.95 (3H, m), 0.95−0.85 (9H, m); 13C NMR (125
MHz, CDCl3) δ 171.0, 170.6, 169.3, 169.1, 156.2, 155.6, 131.6,
131.5,119.0, 118.8, 80.1, 79.8, 74.9, 65.8, 65.6, 64.6, 63.0, 36.5, 30.5,
29.9, 28.3, 27.5, 27.3, 26.1, 26.0, 19.9, 19.8, 19.1, 18.8, 14.3, 14.2, 11.6;
ESIMS m/z 408.2 [M + Na]+; HRMS m/z 408.2352 [M + Na]+ (calcd
for C20H35NNaO6, 408.2362).
CH2Cl2 (30 mL) at 0 °C were added Fmoc-L-Thr(tBu)−OH (970 mg,
2.45 mmol), EDCI (937 mg, 4.89 mmol), and DMAP (598 mg, 4.89
mmol). The reaction was stirred at rt overnight, then diluted with H2O
and extracted with CH2Cl2. The combined organic phase was washed
with brine and dried over Na2SO4. Removal of the solvents followed
by flash chromatography (petroleum ether/EtOAc, 20:1) afforded P3
as a colorless oil (239 mg, 81%): 1H NMR (300 MHz, CDCl3) δ 7.77
(2H, d, J = 7.5 Hz), 7.63 (2H, d, J = 7.4 Hz), 7.41 (2H, t, J = 7.2 Hz),
7.32 (2H, t, J = 7.3 Hz), 5.98−5.83 (1H, m), 5.52 (1H, d, J = 9.3 Hz),
5.36−5.18 (3H, m), 4.57 (2H, d, J = 5.7 Hz), 4.42 (2H, d, J = 6.4 Hz),
4.27 (3H, d, J = 6.3 Hz), 2.19 (2H, s), 1.91 (1H, s), 1.61 (4H, m), 1.20
(15H, s), 1.15 (3H, s); 13C NMR (125 MHz, CDCl3) δ 175.0, 170.6,
156.3, 143.8, 143.7, 141.2, 131.9, 127.6, 126.9, 125.1, 125.0, 119.9,
118.3, 83.6, 77.6, 74.2, 68.8, 67.1, 67.0, 65.4, 60.1, 47.1, 46.7, 29.6,
28.7, 24.9, 22.8, 20.0, 19.2, 17.9; ESIMS m/z 626.4 [M + Na]+; HRMS
m/z 626.3073 [M + Na]+ (calcd for C36H45NNaO7, 626.3094).
(S)-Allyl 2-((S)-1-((6S,9S,12R)-12-sec-Butyl-6,9-diisopropyl-
2,2,8-trimethyl-4,7,10-trioxo-3,11-dioxa-5,8-diazatridecane)-
N-methylpyrrolidine-2-carboxamido)-3-methylbutanoate (12).
To a stirred solution of P2 (380 mg, 1.03 mmol) in CH2Cl2 (10 mL)
at 0 °C was added TFA (2 mL), and the resulting solution was stirred
at 0 °C for 4 h. The reaction mixture was concentrated in vacuo to give
crude intermediate 11. To the solution of 10 (458 mg, 1.03 mmol) in
CH2Cl2 (20 mL) were added HATU (783 mg, 2.06 mmol) and HOAt
(280 mg, 2.06 mmol) followed by addition of the crude intermediate
11 prepared above and DIPEA (0.72 mL, 4.12 mmol). The reaction
was allowed to stir for 14 h and then diluted with H2O. After
extraction with CH2Cl2, the combined organic phase was washed with
brine and dried with Na2SO4. Removal of solvent followed by flash
chromatography (petroleum ether/EtOAc, 3:1) provided 12 as a
colorless oil (429 mg, 60% in two steps): 1H NMR (300 MHz,
CDCl3) δ 5.92−5.76 (1H, m), 5.32−5.16 (3H, m), 5.08−4.99 (2H,
m), 4.94 (1H, d, J = 10.4 Hz), 4.75 (1H, d, J = 8.7 Hz), 4.57 (2H, d, J
= 5.7 Hz), 4.41 (1H, s), 4.04 (1H, s), 3.51 (1H, d, J = 8.4 Hz), 3.03
(4H, s), 2.93 (3H, s), 2.04 (11H, t, J = 56.6 Hz), 1.41 (9H, s), 0.91
(32H, tdd, J = 20.0, 13.8, 6.6 Hz); 13C NMR (100 MHz, CDCl3) δ
172.7, 171.4, 170.7, 169.7, 167.2, 155.7, 131.4, 118.1, 79.0, 75.0, 64.8,
60.8, 60.6, 56.5, 54.8, 46.6, 35.7, 31.2, 30.6, 30.1, 27.1, 26.5, 24.9, 24.1,
19.8, 19.4, 19.3, 18.7, 18.1, 16.9, 14.2, 11.1; ESIMS m/z 717.4 [M +
Na]+; HRMS m/z 717.4434 [M + Na]+ (calcd for C36H62N4NaO9,
717.4414).
(2R,3S)-Allyl-2-((S)-2-((S)-2-(tert-butoxycarbonylamino)-N,3-
dimethylbutanamido)-3-methylbutanoyloxy)-3-methylpenta-
noate (P1). To a stirred solution of 4 (100 mg, 0.26 mmol) in
CH2Cl2 (4 mL) at 0 °C was added TFA (0.6 mL), and the resulting
solution was stirred at 0 °C for 6 h. The reaction mixture was
concentrated in vacuo to give the crude intermediate. To a solution of
N-Boc-L-valine (113 mg, 0.52 mmol) in CH2Cl2 (5 mL) was added
HATU (296 mg, 0.78 mmol) and HOAt (106 mg, 0.78 mmol)
followed by addition of the crude intermediate just prepared and
DIPEA (0.2 mL, 1.04 mmol). The reaction mixture was allowed to stir
for 14 h and then diluted with H2O. After extraction with CH2Cl2, the
combined organic phase was washed with brine and dried with
Na2SO4. Removal of solvent followed by flash chromatography
(petroleum ether/EtOAc, 10:1) provided P1 as a colorless oil (83
1
mg, 66% in two steps): H NMR (300 MHz, CDCl3) δ 5.93−5.79
(1H, m), 5.35−5.22 (2H, m), 5.19 (1H, d, J = 9.5 Hz), 5.07 (1H, d, J =
10.4 Hz), 5.01 (1H, d, J = 3.4 Hz), 4.60 (2H, ddd, J = 5.7, 2.8, 1.4 Hz),
4.43 (1H dd, J = 9.4, 6.4 Hz), 3.06 (3H, s), 2.29−2.17 (1H, m), 2.05−
1.95 (2H, m), 1.82 (1H, d, J = 2.6 Hz), 1.41 (9H, s), 1.33−1.20 (1H,
m), 0.99 (8H, dd, J = 11.0, 6.6 Hz), 0.93−0.79 (15H, m); 13C NMR
(125 MHz, CDCl3) δ 175.1, 172.3, 171.0, 157.9, 133.3, 120.8, 81.3,
77.0, 67.6, 62.8, 57.2, 38.2, 33.3, 32.6, 30.1, 28.7, 27.7, 21.7, 21.4, 20.1,
19.3, 16.1, 13.4; ESIMS m/z 507.2 [M + Na]+; HRMS m/z 485.3223
[M + H]+ (calcd for C25H45N2NaO7, 485.3227).
(S)-Allyl 2-((S)-1-((2R,3S)-2-((S)-2-((S)-2-((S)-3-Hydroxy-2,2-di-
methyloct-7-ynamido)-N,3-dimethylbutanamido)-3-methylbu-
tanoyloxy)-3-methylpentanoyl)-N-methylpyrrolidine-2-car-
boxamido)-3-methylbutanoate (13). The peptide 13 was obtained
from 12 in 63% yield over two steps following a similar procedure to
(S)-tert-Butyl-2-(((S)-1-(allyloxy)-3-methyl-1-oxobutan-2-yl)-
(methyl)carbamoyl)pyrrolidine-1-carboxylate (P2). The frag-
ment P2 (72% yield in two steps) was obtained from 5 following a
1
similar procedure as that for the preparation of the fragment P1. H
1
that for the preparation of the fragment P1: H NMR (300 MHz,
NMR (300 MHz, CDCl3, mixture of rotamers) δ 5.93−5.77 (1H, m),
5.23 (2H, dd, J = 23.8, 13.8 Hz), 4.91 (1H, d, J = 10.4 Hz), 4.68−4.52
(3H, m), 3.64−3.30 (2H, m), 3.00 (3H, d, J = 27.3 Hz), 2.23−1.78
(5H, m), 1.38 (9H, d, J = 7.7 Hz), 0.99 (3H, dd, J = 11.4, 6.6 Hz), 0.89
(3H, dd, J = 6.7, 2.3 Hz); 13C NMR (101 MHz, CDCl3, mixture of
rotamers) δ 173.5, 173.3, 169.5, 169.3, 153.8, 153.7, 131.3, 131.2,
116.9, 116.6, 79.1, 78.8, 64.4, 64.3, 61.7, 56. 7, 56.0, 46.0, 45.7, 29. 9,
29.6, 28.9, 28.2, 26.6, 26.3, 26.0, 23.0, 21.7, 18.2, 17.9; ESIMS m/z
391.1 [M + Na]+; HRMS m/z 391.2192 [M + Na]+ (calcd for
C19H32N2NaO5, 391.2209).
CDCl3) δ 6.63 (1H, d, J = 8.3 Hz), 5.85 (1H, ddd, J = 16.1, 10.6, 5.6
Hz), 5.24 (2H, dd, J = 21.9, 13.8 Hz), 5.07−4.90 (3H, m), 4.80−4.63
(2H, m), 4.57 (2H, d, J = 5.6 Hz), 4.05 (1H, s), 3.55−3.39 (2H, m),
3.03 (3H, s), 2.94 (3H, s), 2.32−2.09 (9H, m), 1.87 (5H, dt, J = 20.0,
8.5 Hz), 1.59 (2H, d, J = 5.7 Hz), 1.34 (2H, dd, J = 19.9, 8.8 Hz), 1.22
(3H, s), 1.17 (3H, s), 1.03−0.76 (24H, m).
(S)-Allyl 2-((S)-1-((5S,8S,12S,15S,18R)-5-((R)-1-tert-Butox-
yethyl)-18-sec-butyl-1-(9H-fluoren-9-yl)-12,15-diisopropyl-
9,9,14-trimethyl-3,6,10,13,16-pentaoxo-8-(pent-4-ynyl)-
2,7,17-trioxa-4,11,14-triazanonadecane)-N-methylpyrrolidine-
2-carboxamido)-3-methylbutanoate (14). To a solution of
peptide P3 (85 mg, 0.14 mmol) in CH2Cl2 (20 mL) at 0 °C were
added Pd(PPh3)4 (16.5 mg, 0.014 mmol) and N-methylaniline (0.045
mL, 0.42 mmol). The reaction was stirred at rt for 4 h. After
evaporation in vacuo, the residue was purified by silica gel
chromatography (petroleum ether/EtOAc, then CH2Cl2/MeOH) to
give the carboxylic acid intermediate as a yellow oil (328 mg, 85%). To
the solution of acid intermediate just prepared (40 mg, 0.071 mmol) in
CH2Cl2 (5 mL) were added HATU (54 mg, 0.142 mmol), HOAt (20
mg, 0.142 mmol), DIPEA (0.05 mL, 0.284 mmol), and the de-Boc
intermediate of 12 (prepared by treating 12 (100 mg, 0.144 mmol)
with TFA (0.5 mL)). The reaction mixture was allowed to stir for 14 h
and then diluted with H2O. After extraction with CH2Cl2, the
combined organic phase was washed with brine and dried with
(S)-Allyl 3-Hydroxy-2,2-dimethyloct-7-ynoate (8). To a
solution of 7 (40 mg, 0.22 mmol) in DMF (3 mL) were added
KHCO3 (44 mg, 0.44 mmol) and allyl bromide (0.028 mL, 0.326
mmol). The reaction mixture was stirred for 4 h at rt and then diluted
with of H2O. The mixture was extracted with diethyl ether, washed
with brine, dried over Na2SO4, and then concentrated under reduced
1
pressure to provide 8 as a colorless oil (46 mg, 93% yield): H NMR
(300 MHz, CDCl3) δ 5.98−5.85 (1H, m), 5.28 (2H, dd, J = 24.3, 13.8
Hz), 4.60 (2H, d, J = 5.6 Hz), 3.64 (1H, d, J = 5.8 Hz), 2.52 (1H, d, J
= 5.7 Hz), 2.24 (2H, td, J = 6.7, 2.6 Hz), 1.94 (1H, t, J = 2.6 Hz),
1.89−1.53 (4H, m), 1.21 (6H, d, J = 5.6 Hz).
Allyl 3-((2S,3R)-2-(((9H-Fluoren-9-yl)methoxy)-
carbonylamino)-3-tert-butoxybutanoyloxy)-2,2-dimethyloct-
7-ynoate (P3). To a solution of ester 8 (110 mg, 0.489 mmol) in
D
dx.doi.org/10.1021/np4001027 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
P1, 5, P2, 6, 7, 8, 9, P3, 12, 13, 14, 1. 1H NMR and 13C NMR
data, comparison of synthetic with natural viequeamide A (1),
and comparison of HPLC for synthetic and natural
viequeamide A (1). These materials are available free of charge
via the Internet at http://pubs.acs.org.
Na2SO4. Removal of solvent followed by flash chromatography
(petroleum ether/EtOAc, 2:1) provided amide 14 as a colorless oil
(429 mg, 68% in two steps): 1H NMR (300 MHz, CDCl3) δ 7.75 (2H,
d, J = 7.3 Hz), 7.61 (2H, d, J = 7.3 Hz), 7.39 (2H, t, J = 7.2 Hz), 7.30
(2H, t, J = 7.2 Hz), 6.65 (1H, d, J = 8.5 Hz), 5.85 (1H, ddd, J = 16.1,
10.7, 5.4 Hz), 5.71 (1H, d, J = 8.9 Hz), 5.30 (1H, d, J = 6.8 Hz), 5.22
(2H, t, J = 9.3 Hz), 5.07−4.90 (3H, m), 4.76 (2H, d, J = 7.6 Hz), 4.57
(2H, d, J = 4.8 Hz), 4.39 (3H, d, J = 7.1 Hz), 4.27 (2H, d, J = 7.5 Hz),
4.07 (1H, s), 3.51 (1H, d, J = 9.0 Hz), 3.03 (3H, s), 2.92 (3H, s),
2.27−1.52 (17H, m), 1.18 (18H, t, J = 8.9 Hz), 1.01−0.71 (38H, m);
13C NMR (100 MHz, CDCl3) δ 177.0, 174.3, 173.6, 172.9, 172.4,
171.7, 169.4, 158.3, 145.8, 145.6, 143.1, 133.6, 129.6, 128.9, 127.0,
121.9, 120.4, 85.6, 80.6, 77.2, 76.2, 70.7, 69.0, 68.9, 67.1, 63.1, 62.9,
62.1, 58.7, 55.7, 49.0, 48.9, 48.5, 38.0, 33.4, 32.8, 32.5, 32.0, 30.6, 30.1,
29.3, 28.6, 27.1, 27.0, 26.3, 25.9, 22.2, 22.0, 21.9, 21.6, 20.9, 20.3, 20.0,
19.3, 16.4, 13.4; ESIMS m/z 1162.5 [M + Na]+.
(3S,6S,9S,13S,16S,19R,24aS)-19-sec-Butyl-6-((R)-1-hydrox-
yethyl)-3,13,16-triisopropyl-2,10,10,15-tetramethyl-9-(pent-4-
ynyl)tetradecahydropyrrolo[2,1-i][1,13,4,7,10,16,19]-dioxa-
pentaazacyclodocosine-1,4,7,11,14,17,20(19H)heptaone (vie-
queamide A, 1). To a solution of peptide 14 (60 mg, 0.053
mmol) in CH2Cl2 (7 mL) at 0 °C were added Pd(PPh3)4 (9 mg, 0.008
mmol) and N-methylaniline (0.017 mL, 0.157 mmol). The reaction
was stirred at rt for 4 h. After evaporation of the solvents in vacuo, the
residue was purified by silica gel chromatography (petroleum ether/
EtOAc, then CH2Cl2/MeOH) to give the carboxylic acid intermediate
as a yellow oil, which was then dissolved in CH3CN (3 mL). To the
solution just prepared, diethylamine (0.6 mL) was added at 0 °C. The
resulting solution was stirred for 1 h and then concentrated in vacuo to
give the N-deprotected intermediate, which was then dissolved in
CH2Cl2 (50 mL). HATU (40 mg, 0.105 mmol), HOAt (14 mg, 0.105
mmol), and DIPEA (0.028 mL, 0.215 mmol) were added sequentially
at 0 °C. The reaction mixture was allowed to stir at rt for 26 h, then
diluted with H2O and extracted with CH2Cl2. The combined organic
phase was washed with brine, dried over Na2SO4, and evaporated. The
residue was purified by column chromatography on silica gel
(petroleum ether/EtOAc, 2:1) to give cyclic peptide 2 (18.7 mg,
41% yield for 3 steps) as a colorless oil: ESIMS m/z 882.8 [M + Na]+;
HRMS m/z 882.5566 [M + Na]+ (calcd for C46H77N5NaO10,
882.5568).
AUTHOR INFORMATION
Corresponding Author
*(A. Zhang) Tel: 86-21-50806035. Fax: 86-21-50806035. E-
mail: aozhang@mail.shcnc.ac.cn. (Y. Xu) E-mail: xuyoujun@
syphu.edu.cn.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants of the Distinguished Young
Scholars of Chinese NSF (81125021), Chinese National
Science Foundation (81072528), and National Science &
Technology Major Project on “Key New Drug Creation and
Manufacturing Program”, China (Numbers 2012ZX09103-101-
035, 2012ZX09301001-001). Supporting grants from Shanghai
Commission of Science and Technology (10JC1417100,
10dz1910104), the “Interdisciplinary Cooperation Team”
Program for Science and Technology Innovation of the
Chinese Academy of Sciences, and the State Key Laboratory
of Medical Neurobiology and Collaborative Innovation Center
for Brain Function and Remodeling (Fudan University) are also
appreciated.
REFERENCES
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D
(c 0.15, CH2Cl2); 1H NMR (300 MHz, CDCl3) δ 8.03 (1H, d, J = 10.4
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2.77 (3H, s), 2.57−2.35 (2H, m), 2.23 (3H, dd, J = 13.6, 6.5 Hz), 2.03
(4H, t, J = 17.2 Hz), 1.93 (1H, t, J = 2.5 Hz), 1.74 (1H, s), 1.68−1.65
(1H, m), 1.56−1.42 (5H, m), 1.36 (3H, s), 1.17 (3H, s), 1.09 (3H, d, J
= 6.7 Hz), 1.01−0.91 (15H, m), 0.86−0.72 (9H, m); 13C NMR (125
MHz, CDCl3) δ 174.7, 172.7, 172.1, 170.3, 169.2, 168.7, 168.2, 84.1,
77.4, 74.5, 68.9, 68.1, 67.4, 63.6, 58.1, 55.7, 53.9, 47.3, 46.6, 36.6, 31.7,
29.6, 29.4, 29.0, 28.2, 27.9, 27.2, 25.9, 25.6, 25.3, 24.7, 20.6, 20.0, 19.5,
19.3, 19.1, 18.8, 18.1, 17.0, 16.1, 13.8, 11.9; ESIMS m/z 804.5 [M +
H]+, 826.6 [M + Na]+; HRMS m/z 804.5103 [M + H]+ (calcd for
C42H70N5O10, 804.5123).
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Cytotoxicity Assay. Cytotoxic effects on the cells were assessed by
sulforhodamine B assays as reported previously.14 The concentration
required for 50% inhibition (IC50) of the tested cells was calculated
using the Logit method.
(13) We thank Prof. William Gerwick and Dr. Paul D. Boudreau,
who originally isolated the natural product viequeamide A, for kindly
providing the authentic sample for our comparison. An HPLC analysis
of natural and synthetic viequeamide A as well as a mixture of both is
included in the Supporting Information.
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ASSOCIATED CONTENT
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S
* Supporting Information
Experimental details for the preparation of compounds 3, 5, 6,
1
7, 9R, 10 and H and 13C NMR spectra for compounds 3, 4,
E
dx.doi.org/10.1021/np4001027 | J. Nat. Prod. XXXX, XXX, XXX−XXX